Epigenetic Regulation in Self-Renewing and Differentiating Male Germ Cells

成年雄性生殖系的维持对于繁殖是必不可少的。仍然是一个知之甚少的过程， 生殖系干细胞的自我更新似乎受到表观遗传的染色质修饰和 随后的核组织。这项建议旨在增加我们对这些问题的理解 表观遗传调控过程。我们假设组蛋白修饰和相关的核蛋白 组成，包括转录抑制复合体和染色质重塑因子，是重要的。 干细胞是自我更新还是分化的决定因素。以小鼠精原细胞为模型，我们 将在两个具体目标中阐述干细胞自我更新的调节。第一个目标将考察 多梳蛋白对自我更新精原细胞基因沉默的影响。为了实现这一目标，有两个截然不同的 精原细胞群体将通过荧光激活细胞分选(FACS)分离并分析 转录特征分析。然后通过染色质鉴定潜在的多梳基团结合位点 免疫沉淀。功能丧失效应将通过以下两种方法之一进行检查：生成 条件基因敲除小鼠或RNAi基因敲除和将培养的精原干细胞移植到 接受者的睾丸。第二个目的是研究核基质蛋白在维持自我更新中的作用。 精原细胞。蛋白质组学分析将在分离核基质蛋白后进行 从FACS分选的生殖细胞中分离，并用液相色谱-质谱仪鉴定其组成。 然后将进行条件基因敲除小鼠或RNAi的生成和移植实验 评估基质相关蛋白的功能重要性。这一奖项的建议研究是 设计为同时包含指导阶段(K99)和独立阶段(Roo)，并进行过渡 计划中包含了一个时期，以成功地将这两个阶段连接起来。这个项目现在正准备进入Roo 阶段，扩大在K99阶段取得的进展，并实现两个具体方面的主要方面 目标。

Maintenance ofthe adult male germline is essential for reproduction. Still a poorly understood process, germline stem cell self-renewal appears to be influenced by epigenetic chromatin modifications and subsequent nuclear organization. This proposal is designed to increase our understanding of these epigenetic regulatory processes. We hypothesize that histone modification and associated nuclear protein composition, including transcriptional repressor complexes and chromatin remodeling factors, are important determinants in whether stem cells self-renew or differentiate. Using mouse spermatogonia as a model, we will address the regulation of stem cell self-renewal in two specific aims. The first aim will examine the role of Polycomb group proteins on gene silencing in self-renewing spermatogonia. To achieve this aim, two distinct spermatogonial populations will be separated by fluorescence-activated cell sorting (FACS) and analyzed by transcriptional profiling. Potential Polycomb group binding sites will then be identified by chromatin immunoprecipitation. Loss-of-function effects will be examined by one of two methods: generation of conditional knockout mice or RNAi knockdown and transplantation of cultured spermatogonial stem cells into recipient testes. The second aim will examine the role of nuclear matrix proteins in the maintenance of selfrenewing spermatogonia. Proteomic profiling will be performed after fractionating nuclear matrix proteins from FACS-sorted germ cells and identifying their composition by liquid chromatography-mass spectrometry. Generation of conditional knockout mice or RNAi and transplantation experiments will then be performed to assess the functional importance of matrix-associated proteins. The proposed research for this award is designed to encompass both the mentored phase (K99) and the independent phase (ROO), with a transition period built into the plan to successfully bridge the two phases. This project is now poised to enter the ROO phase, extending the progress made during the K99 phase and achieving the major aspects of both specific aims.